Transmucosal pentobarbital is a successful euthanasia method in pond slider turtles (Trachemys scripta)

Zachary C. Ready Wildlife Epidemiology Laboratory, College of Veterinary Medicine, University of Illinois, Urbana, IL
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL

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Krista Keller Wildlife Epidemiology Laboratory, College of Veterinary Medicine, University of Illinois, Urbana, IL
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL

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Abstract

OBJECTIVE

To determine the ability of transmucosal euthanasia solution to induce euthanasia in pond slider turtles (Trachemys scripta).

ANIMALS

16 pond slider turtles (T. scripta).

PROCEDURES

Pentobarbital 100 mg/kg was delivered through esophageal gavage (n = 8) or cloacal administration (8). The presence of voluntary movement, heart rate (HR), respiratory rate (RR), palpebral reflex, corneal reflex, and response to noxious stimuli were recorded until death, confirmed via the absence of reflexes, movement, heartbeat, and absence of cardiac electrical activity.

RESULTS

No signs of irritation were observed in any turtles. Leakage after administration occurred in 75% (6/8) of the cloacal group, including 2 turtles with marked leakage or expulsion. Two of eight turtles in the cloacal group regained movement and required euthanasia by a standard method and 1 turtle in the oral group was excluded from further analysis due to a miscalculated dose. The remaining 13 turtles (7/8 oral, 6/8 cloacal) had cessation of a heartbeat at a median of 18 hours (range = 6 to 26 hours) with respiratory arrest occurring within 15 minutes. The median time to loss of corneal reflex was 45 minutes (range = 15 minutes to 4 hours). Time-to-loss of parameters was similar between oral and cloacal routes, respectively.

CLINICAL RELEVANCE

Transmucosally administered pentobarbital via the oral and cloacal routes both result in euthanasia within approximately 24 hours. Given that 25% of the turtles in the cloacal group required a secondary method of euthanasia, the oral route is a preferred route to induce euthanasia in pond turtles.

Abstract

OBJECTIVE

To determine the ability of transmucosal euthanasia solution to induce euthanasia in pond slider turtles (Trachemys scripta).

ANIMALS

16 pond slider turtles (T. scripta).

PROCEDURES

Pentobarbital 100 mg/kg was delivered through esophageal gavage (n = 8) or cloacal administration (8). The presence of voluntary movement, heart rate (HR), respiratory rate (RR), palpebral reflex, corneal reflex, and response to noxious stimuli were recorded until death, confirmed via the absence of reflexes, movement, heartbeat, and absence of cardiac electrical activity.

RESULTS

No signs of irritation were observed in any turtles. Leakage after administration occurred in 75% (6/8) of the cloacal group, including 2 turtles with marked leakage or expulsion. Two of eight turtles in the cloacal group regained movement and required euthanasia by a standard method and 1 turtle in the oral group was excluded from further analysis due to a miscalculated dose. The remaining 13 turtles (7/8 oral, 6/8 cloacal) had cessation of a heartbeat at a median of 18 hours (range = 6 to 26 hours) with respiratory arrest occurring within 15 minutes. The median time to loss of corneal reflex was 45 minutes (range = 15 minutes to 4 hours). Time-to-loss of parameters was similar between oral and cloacal routes, respectively.

CLINICAL RELEVANCE

Transmucosally administered pentobarbital via the oral and cloacal routes both result in euthanasia within approximately 24 hours. Given that 25% of the turtles in the cloacal group required a secondary method of euthanasia, the oral route is a preferred route to induce euthanasia in pond turtles.

According to the most recent survey conducted by the AVMA, 3.7 million households across the United States have at least 1 companion reptile; this represents a 23.3% increase from 3.0 million households owning reptiles as pets in 2012.1 A rapid rise in reptile ownership underscores the importance of implementing humane and acceptable euthanasia methods for companion reptiles that do not jeopardize the human-animal bond. Further, reptiles are kept in zoological institutions, in laboratory settings and are important species within their free-ranging ecosystems. Various challenges are encountered in the euthanasia of reptiles, including IV access, temperature-dependent drug metabolism, variable effects of inhalants due to sustained breath-holding or intracardiac shunting, and capacity for anaerobic metabolism.4,8,11 This represents a need for alternative methods with easier access, reliable induction of unconsciousness, and minimal distress to the animal.

The AVMA’s current guidelines for acceptable methods of euthanasia in individual reptiles primarily recommend the administration of barbiturates, namely sodium pentobarbital, with the intravascular route preferred.2 Sodium pentobarbital is the primary active ingredient in many euthanasia solutions available to clinicians, with sodium phenytoin, a cardiovascular depressant, the second active ingredient.14 Dissociative agents, inhaled agents, IV anesthetics, and other short-acting barbiturates may be used alone or in combination to induce a general anesthetized state and subsequent euthanasia.2 An adjunctive method is recommended, which may include pithing as part of a 2-step euthanasia protocol, or decapitation as part of a 3-step protocol. Unfortunately, these physical adjunctive methods are unpalatable to the public and may threaten the human-animal bond and trust in the veterinary community when pursued in the companion sector.

Given these challenges, the authors of this prospective study investigated the effects of transmucosally administered euthanasia solution on a common species of aquatic chelonian (Trachemys scripta). It was hypothesized that transmucosal pentobarbital would induce euthanasia without the need for a secondary method over a period of 24.0 hours (oral) or 48.0 hours (cloacal).

Materials and Methods

Animals

The study protocol was approved by the University of Illinois Institutional Animal Care and Use Committee (Protocol #21231). Sixteen slider turtles (T. scripta) of undescribed sex were included; 14 of which were red-eared sliders (Trachemys scripta elegans). The turtles were originally sourced from a reptile rescue for an infectious disease trial but did not qualify for the study and were destined for euthanasia.

Husbandry

The turtles were maintained in groups of 2 in 50-gallon opaque sided open-topped plastic enclosures (101.0 cm X 54.6 cm X 45.4 cm; Sterilite Corporation), filled ∼60% with fresh water, and a haul-out space was provided. Tank water was emptied and refilled 3 times weekly. During the entire experiment and for the 4 weeks before, the turtles remained in a temperature-controlled environment with air temperatures of 27.8 °C (82.0 °F), which is within the preferred optimal temperature zone (POTZ) of this species.16 Humidity was maintained at >60%. Full spectrum lighting was provided on a 12-hour light cycle using fluorescent tube lights. Turtles were offered a variety of leafy greens including kale and red leaf lettuce every other day and a commercial floating turtle pellet every other day (Reptomin, Tetra, Spectrum Brands Pet, LLC). For 24 hours before the study, food was withheld.

Administration of euthanasia solution

Two hours before euthanasia, the water and haul-out components of the habitats were removed so that the turtles were maintained in a dry, plastic tub in pairs. Baseline measurements were obtained including heart rate (HR; measured via Doppler ultrasonic flow detector in the cervical window), respiratory rate (RR; via observation of body wall movements at the cranial aspect of the body), and ambient temperature and humidity. The turtles were randomly assigned (n = 8 to each group) via a random number generator to receive pentobarbital transmucosally via the oral mucosa (ORAL) or the cloacal mucosa (CLOACAL). Pentobarbital sodium and phenytoin sodium (EUTHASOL® Virbac AH, Inc, 39% pentobarbital, 5% phenytoin sodium; 100 mg/kg dose per pentobarbital concentration) was administered transmucosally either the oral or cloacal route after rounding to the nearest 0.1 mL volume. Oral administration was accomplished by manually restraining the head with the extension of the neck with the turtle held in sternal recumbency or upright, then the beak was opened using a plastic surgical nail pick if the turtle did not voluntarily open their mouth and an 18 g stainless steel feeding tube was used to deposit the pentobarbital over the esophageal mucosa at the level just cranial to the thoracic inlet. Confirmation of the feeding tube in the esophagus was performed by palpating the metal tube in the esophagus separately from the trachea and pressing the end of the metal bulb against the skin at the level of the thoracic inlet to create a visible bulge at this site. After dispensing the pentobarbital in the syringe, 1 mL of air was used to push any remaining solution over the mucosa. The metal feeding tube was withdrawn slowly, and the oropharynx was observed for any reflux of solution or visible leakage. The turtle was then placed back in a clean and dry container for serial observations to be made, atop a white paper towel to monitor for regurgitation of solution.

Cloacal administration was accomplished by extending the tail with the turtle in dorsal recumbency or held upright, and the ball of an 18 g stainless steel feeding tube was introduced into the vent opening and advanced 2–3.0 cm into the cloaca without resistance. The pentobarbital was delivered over a 1–2-minute time frame and 0.5 mL of air was used to flush any remaining solution into the cloaca. The tube was then withdrawn slowly to prevent backflow and the vent was observed for any reflux of pentobarbital solution or excretion of urine or feces. The turtle was then placed back in a clean and dry container for observations to be made. Because the solution used was pink, a white paper towel was placed underneath the turtles that had pentobarbital administered via the cloaca to facilitate observation of leakage from the administration site.

Observations

After the completion of euthanasia solution administration, subjective and objective parameters were recorded until confirmation of death. Subjective observations recorded included the presence of or absence of voluntary movement without stimulus, voluntary movement with stimulus, any visible “leakage” of pentobarbital from the administration site, clawing at the mouth or excessive yawning (oral) or kicking hind limbs at the tail (cloacal). Objective observations recorded included RR, HR, palpebral reflex, corneal reflex, and response to noxious stimuli-superficial pain and deep pain. Observations were made starting 15 minutes after administration and every 15 minutes thereafter (first 60 minutes), then every 30 minutes (next 2 hours), then every 60 minutes (next 4 hours), and then every 4 hours thereafter.

Leakage from the administration site was defined as any solution that leaked from the oral cavity or any pink discharge, consistent with the color of the euthanasia solution, from the vent after administration. Voluntary movement without stimulus was defined as present if any visible movements were made by the turtle before handling or physical stimulation. Voluntary movement with the stimulus was defined as present if visible movement occurred during handling, including tactile stimulus of the head, thoracic limbs, and pelvic limbs, and positioning of the Doppler probe for the recording of the HR.

RR was measured by direct visualization of respiratory movements performed before handling and counting over 60 seconds; if no respiratory movements occurred for 60 seconds, the observer would record for 120 seconds. The HR was measured by placing a Doppler probe in the cervical window (between the thoracic limb and neck) and monitoring for flow at the level of the heart. HR was counted over 60 seconds, if no HR was detected then monitoring would continue for 5 minutes. The presence or absence of arrhythmias was recorded. Positive response for palpebral reflex was defined as the movement of the superior or inferior palpebrae, or nictitating membrane in response to using a cotton-tipped applicator (CTA) to tap on the medial and lateral canthi of the eye. If the palpebral reflex was absent, the corneal reflex was tested on the same eye. Positive response for the corneal reflex was defined by globe retraction that occurred in response to using a moistened (saline) CTA to gently touch the cornea. For both palpebral and corneal reflexes, the eye tested (left eye or right eye) was randomized for each observation at each time point. Response to noxious stimuli was evaluated via response to superficial pain, and response to deep pain was assessed if superficial pain was absent. Response to superficial pain was defined as positive if the limb was withdrawn in response to a skin pinch applied by a hemostat to the distal aspect of the thoracic limb and ipsilateral pelvic limb. Lack of response was defined as no reaction occurring after allowing the pinch to continue for 5 seconds. Randomization was applied to assign whether the left or right limbs were assessed, and in which order (thoracic limb first and pelvic limb second, or vice versa). Deep pain response was assessed on the same limbs as the superficial pain was assessed, in the same randomized order. Deep pain response was defined as positive if the limb was withdrawn in response to the application of a firm pinch by a hemostat over the metacarpal or metatarsal bones. The pinch was allowed to stay on for 5 seconds, but no longer, if no response was documented. The turtles were housed in a container so that if they were to recover, they would not be able to climb or injure themselves.

Confirmation of death

Once there was the absence of detectable HR, RR, voluntary movement, reflexes, and response to noxious stimuli, electrocardiogram (ECG) leads were attached using the standard for Lead II positioning for electrocardiography. If no electrical conduction was present for a period of at least 5 minutes, death was confirmed at that time.

If turtles did not respond to the initial administration of transmucosal pentobarbital (maintained movement, and reflexes for greater than 10 hours after initial dosing), they were euthanized by acceptable methods (pentobarbital 100 mg/kg IV).2

Statistical analysis

The times-to-loss of voluntary movement (when stimulated or not stimulated), HR, RR, palpebral reflex, corneal reflex, superficial pain, and deep pain were recorded for each turtle; descriptive statistics were calculated for the times-to-loss of all subjective and objective parameters observed, and parameters were compared between oral and cloacal groups using a Mann-Whitney test to account for small sample sizes, with a significance of P ≤ .05. All statistical analyses were performed using commercially available software (Prism 9 for macOS, Version 9.3.1, Graphpad Software).

Results

Body weight ranged from 0.19–0.73 kg (0.40–1.61 lb) with a median value of 0.30 kg (0.66 lb). The median straight carapace length (notch to notch) was 12.0 cm (range = 10.0 to 19.0 cm). The median straight carapace width was 10.0 cm (range = 7.9 to 14.0 cm). The median curved carapace length was 13.0 cm (range = 11.0 to 20.0 cm) and the median curved carapace width was 12.0 cm (range = 9.9 to 18.5 cm).

The total volume of euthanasia solution delivered ranged from 0.1 to 0.3 mL (median 0.1 mL, mean 0.2 mL). Because of the methodology used that included rounding euthanasia solution volumes to the nearest 0.1 mL, pentobarbital doses administered ranged from 103.0 mg/kg to 205.0 mg/kg, with a mean dosage received of 138.3 mg/kg, excluding a single turtle that was administered an overdose (664.8 mg/kg) due to a calculation error. The dose of phenytoin delivered to the turtles ranged from 13.2–26.2 mg/kg with an average dose of 17.8 mg/kg, excluding the overdosed turtle who received 85.2 mg/kg. No turtles showed signs indicating irritation secondary to transmucosal administration of euthanasia solution (clawing at the mouth or excessive yawning [oral] or kicking hind limbs at tail [cloacal]). For the cloacal group, leakage of the euthanasia solution after administration occurred to varying degrees in 6/8 turtles and was classified as minimal (n = 1), mild (n = 3), or marked (n = 2).

Three turtles were excluded from further descriptive and statistical analysis of time-to-loss of functional parameters before death: 1 turtle received a cloacal dose of euthanasia solution and was actively ambulating and had maintained all reflexes and normal parameters by 14 hours post-initial administration. No leakage from the cloaca was documented after administration. RR was initially 4 breaths/min before administration and decreased to 0–2 breaths/minute after administration. No respirations were seen at 1 hour to 2 hours post-administration, but respirations resumed thereafter. At 4–14 hours post-administration this turtle was noted to be walking around and displaying normal behaviors during handling. HR remained stable at 60–68 bpm until 6 hours post-initial administration; when it decreased to 50 bpm and then decreased again to 36 bpm at 10 hours post-initial administration. However, the turtle was still physically and behaviorally active at this time. At 14 hours post-initial administration the turtle was euthanized using a standard method (100 mg/kg pentobarbital IV) and excluded from the descriptive and statistical analysis.

A second excluded turtle had mild leakage of euthanasia solution from the vent after cloacal administration. Voluntary movement, spontaneous respiration, and palpebral reflex were lost at 15 minutes post-administration; however, all were regained at 4 hours post-administration. Corneal reflex was absent at 30-minute, 1-hour, and 2-hour timepoints but otherwise maintained. No observable trend in HR occurred over time (HR ranged from 36–55 bpm) with the lowest reading occurring at 10 hours post-administration. Behaviorally, the turtle was noted to be stumbling around in the enclosure between 4–14 hours post-administration. At 14 hours, this turtle was ambulating in an uncoordinated fashion and exhibiting head-pressing behavior and due to poor response, was euthanized using a standard method at 14 hours (100 mg/kg pentobarbital intravenously) and excluded from the remainder of the analysis. Head-pressing behavior was assumed to be secondary to the sedative effects of pentobarbital administration.

One turtle receiving solution via the oral route had an accidental overdose due to a miscalculation that was over 3 times the intended amount; therefore, this turtle was excluded from the remainder of the analysis. This turtle had death confirmed 14 hours post-administration. Loss of voluntary movement, superficial pain, deep pain, palpebral reflex, and spontaneous respirations occurred 15 minutes post-administration. Loss of corneal reflex occurred 30 minutes post-administration. HR did not follow a linear decline and fluctuated from 15–35 bpm until it had decreased by 50% at 6 hours and continued to decline until death was confirmed at 14 hours.

For all remaining 13 turtles (6 cloacal and 7 oral), cessation of voluntary movement without stimulus was documented in 15 minutes or less with no significant difference between oral and cloacal groups (P = .99; Figure 1). Cessation of voluntary movement with the stimulus was documented within 15 minutes for 69.2% (9/13) of turtles and within 30 minutes for 92.3% (12/13) of turtles. There was no significant difference between the 2 groups for a cessation of voluntary movement with the stimulus (P = .22). Loss of spontaneous respirations was documented in 15 minutes or less for all turtles, with no statistical differences in time-to-loss between groups (P = .46).

Figure 1
Figure 1

Individual data scatter plots highlighting median values for time-to-loss of voluntary movement without stimulation (A), voluntary movement with stimulation (B), heart rate (C), respiratory rate (D), palpebral reflex (E), corneal reflex (F), superficial pain (G), and deep pain (H) after administration of transmucosal euthanasia solution to pond slider turtles (Trachemys scripta) via esophageal gavage (n = 7) or cloacal administration (n = 6). There were no statistical differences in any parameters based upon nonparametric comparisons (P value range .17–.99).

Citation: American Journal of Veterinary Research 84, 7; 10.2460/ajvr.23.01.0011

Median time to 50% decrease in HR occurred at 6 hours for both groups (oral: range = 0.5–10.0 hours, cloacal: range = 1.5–14.0 hours), and median time to death after a 50% decrease in HR occurred within 12 hours for both groups. Cardiac bradyarrhythmias were documented in 61.5% (8/13) of turtles at a median time of 10 hours post-administration (range = 4–24.5 hours), the arrhythmias were not further investigated with ECG. Arrhythmias were noted in 71.4% (5/7) of the oral group and 50% (3/6) of the cloacal group. Electrocardiography confirmed asystole in all turtles. One of the turtles in the oral group had pulseless electrical activity observed on ECG at 22 hours post-administration, and asystole was confirmed on ECG at 26 hours, otherwise time to death was the same as the time to arrest of HR for all other turtles. There was no difference between the time to cessation of HR between groups (P = .89; Figure 1).

Loss of palpebral reflexes occurred in 30 minutes or less for 61.5% (8/13) of turtles and 45 minutes or less for 76.9% (10/13) of turtles. The median time-to-loss of palpebral reflexes was 30 minutes (range = 15 minutes to 3 hours) for all turtles, without a difference noted between groups (P = .91; Figure 1). The median time-to-loss of corneal reflexes was 30 minutes (range = 15 minutes to 3 hours). Time-to-loss of palpebral reflex and corneal reflex was documented simultaneously in 9/13 (69.2%) of turtles, either at 30 minutes or 45 minutes post-administration. For the remaining 4 turtles, loss of corneal reflex was documented within 1 hour or less after loss of palpebral reflex. There was no difference in time-to-loss of corneal reflex between groups (P = .56).

The median time-to-loss of superficial pain was 15 minutes (range = 15 minutes to 1.5 hours), with loss of response to superficial pain occurring in 30 minutes or less for 76.9% (10/13) of turtles. The median time-to-loss of deep pain was 15 minutes (range = 15 minutes to 1.5 hours). Loss of superficial and deep pain response occurred simultaneously and within 45 minutes or less for 92.3% (12/13) of turtles; the remaining turtle had experienced mild leakage after administration and lost deep pain at 1.5 hours post-administration, 15 minutes after losing superficial pain. There were no differences in time-to-loss of superficial (P > .54) or deep pain (P = .31) between groups (Figure 1).

The median time to death was 18 hours (range = 6–26 hours) for all turtles, with no significant difference between the oral and cloacal administration groups (P = .99).

Discussion

Euthanasia is the practice of ending the life of a patient in a way that minimizes or eliminates pain and distress.2 Techniques used should be pain-free, resulting in a rapid loss of consciousness followed by cardiac or respiratory arrest and, ultimately, loss of brain function.5 Transmucosal administration of euthanasia solution was a successful stand-alone method of euthanasia for all turtles in the oral group and 75% (6/8) of the turtles in the cloacal group, without the need for a secondary method. Both the oral and cloacal routes of administration accomplished euthanasia with minimal differences noted in the time-to-loss of observed parameters.

Head and limb retraction into the shell frequently prohibits access to common IV injection sites. While the subcarapacial sinus is accessible, inadvertent intrathecal injection or injection into the lymphatics is a considerable risk for this site, reducing the likelihood of immediate bioavailability.13 Furthermore, extravasation of the drug into the lung, bronchi, or trachea can occur at this injection location.15 Chelonians’ capacity for breath holding and intracardiac right-to-left shunting can result in a lack of clinical effect from inhalants, and the slow metabolism of parenteral agents can delay the procedure.4,8,11 Although the head or tail must be able to be restrained for administration, transmucosal pentobarbital in chelonians provides a potential alternative that does not require inhalant anesthetics or access to IV sites in conscious animals.

Given that 2 out of 8 turtles in the cloacal administration group required an injectable euthanasia method due to return of consciousness, and that some degree of leakage or expulsion of the drug occurred in 75% (6/8) of the turtles with cloacal administration, this route should be considered less reliable. Esophageal drug absorption may have been more reliable than the cloacal route for multiple reasons. Once delivered into the esophagus, the risk of regurgitation or leakage of the oral drug was minimal and was not noted in the turtles with our methodology. Cloacal delivery likely resulted in more variable absorption as insertion of the metal feeding tube may have stimulated the desire to urinate or prolapse the phallus, inadvertently resulting in loss of drug deposited. Furthermore, the drug could have been retropulsed in the urinary bladder or colon, potentially resulting in different rates of absorption. The authors determined “cloaca” to be the most appropriate term for this delivery method, given that the method described may result in drug deposition over mucous membranes in up to 3 locations: the colon, urinary bladder, and/or cloaca.10 Further, drugs administered in the caudal half of the body of reptiles may be routed through the renal or hepatic portal systems, decreasing bioavailability.7

Pentobarbital is absorbed rapidly from the gut after oral or rectal administration in mammals.14 Onset of action occurs within 15–60 minutes of oral dosing and 1 minute after IV administration and distributes rapidly to all body tissues.14 Phenytoin sodium is the second active ingredient in the euthanasia solution used, added for its cardiac depressant effects.14 Like pentobarbital, it is also absorbed orally, with cloacal/rectal absorption not evaluated.14 Both ingredients are metabolized by the liver and excreted in the urine.12 It is unknown what the mucosal or gastrointestinal bioavailability of either drug is in turtles, and evaluating this was outside the scope of the study. However, it is reasonable to suggest that turtles that had the drug delivered within the esophagus may have had variable entry of the drug into the stomach. No method was used to determine if the drug passed into the stomach, which was a limitation of this study.

In the 2 turtles that were excluded from the analysis due to return of movement, the administered cloacal dose may have been partially expelled, incompletely absorbed, or metabolized through hepatic or renal portal pathways, resulting in initial sedative effects then at least partial return of a function (which occurred at 4–14 hours). This demonstrates that if dosed or delivered incompletely in turtles, transmucosal administration of euthanasia solution may not achieve the desired effect of euthanasia and these turtles may regain consciousness. One animal was ambulating and head pressing, which was likely due to a lack of coordination due to residual sedation from drug administration. For this reason, monitoring of the patient between 4 and 14 hours post-administration may be advised and a traditional method may be recommended if euthanasia solution is delivered via cloacal administration and desired clinical effect is not seen within that time frame. None of the turtles delivered oral transmucosal pentobarbital regained consciousness in this study.

While the AVMA guidelines for euthanasia require a rapid loss of consciousness, acceptable timeframes for euthanasia in reptiles are poorly defined.2 Additionally, the loss of consciousness is challenging to define in reptiles and lack of response to noxious stimuli often defines this timepoint. The median time-to-loss of superficial and deep pain responses was 15 minutes, which is comparable to expectations of a loss of consciousness after the administration of sedation or anesthetic drugs in this taxon. Further, our methodology allowed the first assessment of observations at the 15-minute time point; given most turtles in this study had lost superficial and deep pain at this time point it is unknown at which specific minute after euthanasia solution administration that consciousness was lost.

In the turtles presented here, it is unknown if cerebral death preceded the timepoint defined as “death,” given that reptiles have intrinsic myocardial activity independent of central nervous system input.12 Cessation of heartbeat is often an unreliable way of defining death in ectothermic species with intrinsic myocardial activity, which makes confirmation of death challenging.12 It is unknown at which point in the 26 hours of monitoring, each turtle could be assigned as cerebral death and future studies should assess cerebral death as an outcome. All turtles in this study were kept in a climate-controlled room, within the species’ POTZ to ensure that drug absorption and distribution were optimized.3,8 In clinical settings, this is not a reasonable expectation to have a euthanized patient be maintained in a clinic or brought home with external heat for 24 hours. Further studies are necessary to determine how long heat must be provided to support absorption and drug distribution to limit the time necessary for post-administration heat support.

Most turtles achieved a deep plane of general anesthesia (defined as a lack of palpebral/corneal reflexes, voluntary movement, and response to noxious stimuli) in 45 minutes or less. Therefore, transmucosal pentobarbital may also be used in the clinical setting as a novel first-step technique as part of a 2-step euthanasia protocol. Once the turtle is deeply anesthetized via transmucosal pentobarbital administration, clinicians would have the opportunity to perform a secondary method, such as IV injection (ie, with potassium chloride or pentobarbital).2

The sequence of events leading to death after administration of euthanasia solution described for mammals begins with rapid induction of unconsciousness progressing to deep anesthesia and concomitant reduction in blood pressure.5 Depression of the medullary respiratory center results in respiratory arrest, cerebral death is indicated by isoelectric activity on encephalography, and cardiac activity then ceases.5 Use of electroencephalography was outside the scope of this study; however, loss of physiologic parameters reflective of central nervous system function occurred in this predicted sequence of events. The same sequence of loss of physiologic parameters was observed for all turtles, allowing the observer to determine which parameter was due to be lost next in the sequence. Voluntary movement without stimulus, voluntary movement with stimulus, and spontaneous respirations were always the first parameters to be lost (at 15 minutes or less). These were followed by loss of superficial pain and deep pain (median 15 minutes), palpebral reflex (median 30 minutes), and corneal reflex (median 45 minutes). Respiratory arrest preceded cardiac arrest by 14–26 hours in the turtles in this study, which is consistent with the predicted sequence of events after the administration of euthanasia solution in mammals.5

In leopard geckos (Eublepharus macularis), euthanized via intraceolomic (ICe) or intracardiac (ICa) injections of pentobarbital at 800 mg/kg, loss of righting reflex occurred first, followed by loss of response to noxious stimuli and then cessation of heartbeat.9 The doses used in these lizards were significantly higher than in our study; however, this publication was not available at the time that our study was performed. The time to the cessation of heartbeat for geckos administered ICe and ICa pentobarbital was 35 minutes and 0 minutes, respectively, a much faster time to arrest than the turtles in our study.9 It is possible that increasing the dose of transmucosal pentobarbital to 800 mg/kg in the turtles would have achieved faster results; however, the volume of euthanasia solution required would be increased. The disadvantage to the ICe and ICa methods was that the geckos receiving pentobarbital arched their backs, suggesting irritation, and animals euthanized via ICa injection had artifactual changes in histopathology.9

Despite the lack of obvious adverse effects noted in the turtles here, the authors were concerned about irritation or corrosion of mucosal surfaces, given that the pH of the euthanasia solution used is 12–13.5 Adverse effects associated with oral transmucosal administration of euthanasia solution has been reported in 27.3% of birds and were often excitatory in nature including tremors, head shaking, wing flapping, and seizure-like activity.6 It is unknown if these effects were mediated by mucosal damage or the excitatory phase of administered barbiturates. While histopathology was not performed in the turtles in this study to rule out histopathologic changes, the absence of clinical signs related to irritation associated with the drug supports this may be a non-painful, humane procedure. While 1 turtle who initially became anesthetized, then re-awoke was head pressing, the authors hypothesize this was due to clinical central nervous system effects from the pentobarbital, and less likely to be a conscious pain response. Pentobarbital has been shown to cause clinically significant artifacts on histopathology in ICa injections in leopard geckos (but did not produce histopathologic changes after ICe administration).9 Despite this, it may be advisable to avoid this technique in animals with alimentary tract disease where post-mortem evaluation is desired until further research is performed.

This study evaluated transmucosal euthanasia solution at a single dose, which because of rounding became a dose range of 100.0–200.0 mg/kg of pentobarbital. The calculated dose of pentobarbital at 100.0 mg/kg was selected based on the standard 1 mL/10 lb recommendation in domestic small animal medicine. In another study in wild birds, much higher doses of pentobarbital were administered transmucosally (430.0–602.0 mg/kg) to induce euthanasia.6 Most birds in this study achieved a rapid loss of consciousness, respiratory arrest, and asystole within 5 minutes.6 Further, the turtle in this study that was administered the highest dose (664.0 mg/kg) and excluded from the data analysis showed a more rapid onset of all observations compared with the median of the other turtles administered oral euthanasia solution. It is unknown if the differences noted represent purely taxa differences or drug dose differences but highlights opportunities for future work to include higher doses of pentobarbital in turtles and other reptile species. Future investigations are encouraged to compare this method described to more traditional 2- and 3-step euthanasia techniques as recommended by the AVMA guidelines for euthanasia.2

The method described here may be especially useful for cases where IV, ICa, or IP injection of euthanasia solution is impossible and where a prolonged period before death is acceptable, such as in wildlife cases. Biologists who need to euthanize their subjects for research may find this method suitable, given that this is a single-product euthanasia that does not require the use of sharps.

In summary, transmucosal administered pentobarbital resulted in humane euthanasia in pond slider turtles without the need for a secondary method when administered orally. It was also successful when administered cloacally in most cases. In successful cases, our methods resulted in a smooth loss of consciousness progressing to a general anesthetized state within 45 minutes or less, and death was confirmed for most cases in 24 hours or less when the turtles were kept within their POTZ. Bradyarrhythmias are a common side effect to be expected if monitoring HR.

Acknowledgments

The authors have nothing to declare.

The authors thank the students that contributed to the care and well-being of the turtles used in this study: Maris Daleo, Kelly Loeb, Maural Sowlat, and Caroline Terry.

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